HIV-1 is the causative agent of AIDS. Three viral enzymes -- reverse transcriptase (RT), integrase (IN), and protease (PR) -- have essential roles in the replication of HIV-1. We are engaged in a long-term effort to study HIV-1 RT, with the expectation that this information will be useful in the development of more effective anti-RT drugs. Our strategy has involved the analysis of both wild-type and mutant HIV-1 RTs, including drug-resistant mutants. Some of this purified RT has been used by our long-term collaborator, Dr. Eddy Arnold (Rutgers University), for structural studies. We have used purified HIV-1 RT to study the biochemical properties of RT mutants, including drug-resistant mutants. There are two clinically important classes of inhibitors of HIV-1 RT: nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). Both are used to treat HIV-1 infections; however, there are serious problems with drug toxicity and with the development of resistance. A major focus of our work on HIV-1 RT is the mechanism(s) of RT inhibitor resistance. (a) NRTIs and NRTI resistance. We are continuing to investigate the mechanisms of resistance to NRTIs, using both structural analysis and biochemical assays. We are also interested in elucidating the mechanism of action of the unconventional chain terminators (UCTs). Ongoing structural analysis in Dr. Arnold's group should shed light on the underlying mechanism(s). We are engaged in collaborative efforts to determine the toxicity of the UCTs.(b) Developing new NNRTIs. We are generating and testing new NNRTIs. We have several promising compounds that are able to inhibit wild-type (WT) and several common NNRTI-resistant viruses with IC50s below 5 nM. These compounds have CC50s for cells in culture more than 4 logs higher than their IC50s. Additional compounds are being designed and synthesized based on the structures/properties of the most promising compounds, modeling, and X-ray structures (generated by Dr. Arnold's group) of the compounds in complexes with WT and drug-resistant RTs. (c) Mutations that affect RT stability in virions. We are studying the effects of mutations in RT and in the HIV-1 genome on reverse transcription. We showed that a large percentage of mutations in the thumb subdomain make RT susceptible to PR cleavage; in some cases, this susceptibility creates a temperature-sensitive (ts) phenotype. RT degradation can affect viral fitness; we will look at additional mutations outside the thumb. We recently showed that the purified RTs are ts, and we have developed E. coli expression systems to investigate the folding and proteolytic sensitivity of these mutant RTs. We will determine whether the effects of the mutations reside in the p66 subunit, the p51 subunit, or both.(d) Fidelity of HIV-1 replication in cell culture. We used a lacZalpha complementation assay similar to the assays used by the Pathak and Mansky laboratories to measure the fidelity of HIV-1 replication in vitro. We have isolated and sequenced a sufficiently large number of mutations to identify hotspots and determine the pattern of lacZalpha mutations for WT RT and a number of mutants, including drug-resistant mutants. The data show that (1) all of the published in vitro assays using purified RT overestimate the in vivo error rate and fail to correctly identify the mutational hotspots; (2) which strand of lacZalpha is in the RNA genome does not affect the overall error rate, or the types of errors made, but it does affect the hotspots; (3) based on preliminary data, mutations in RT affect which sites are mutational hotspots; (4) in our cell culture system, Adenine Deaminase RNA-Specific (ADAR) may cause multiple mutations; (5) all of the RT mutants analyzed so far have mutation rates higher than WT.(e) Reverse transcription complexes (RTCs). We have been working on the development of protocols to isolate RTCs from infected cells to determine the host and viral proteins present in the RTCs. Although progress has been made, we were not able to produce sufficient quantities of material to identify the proteins present in RTCs and are therefore not pursuing this project.[Corresponds to Hughes Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]